Over the last decades, in vivo fluorescent imaging has emerged as a powerful tool in biological studies, owing to its potential to visualize the dynamic processes of an organism development. In order to enable the technology, one has to find a practical biological imaging agent, or bioimaging agent, that can constantly recognize and track the specific biological target. In a typical process, fluorescent materials are linked to an antibody, protein, or mRNA to achieve good targetability, which often involves tedious procedures and high costs.
In recent years, the zebrafish (Danio rerio) has become a favorite model organism for studying vertebrate development, due to its prolific reproduction, transparency, and high homology with mammals. In the embryonic development of all teleost fishes, a remarkable feature is the formation of the yolk syncytial layer (YSL), an extra-embryonic tissue occurring at the surface of the yolk cell. YSL plays crucial roles in embryo patterning and morphogenesis, such as specifying mesoderm and endoderm cell fates in marginal blastomeres along the circumference of the embryo. It is also involved in the regulation of heart progenitor cell migration and essential metabolic functions.
In prior art YSL-imaging methods, non-diffusible fluorescent dyes (attached to either dextran or proteins) are injected into the top of the yolk close to yolk/blastoderm margin, where YSL is expected to develop. The injection process is normally carried out immediately before the YSL is formed. Since the YSL itself is a highly dynamic tissue undergoing extensive movements of overlying germ layer progenitor cells, it is very difficult to achieve accurate injection that can label the entire YSL region. The stability, targetability, and reliability of a fluorescent signal by using the above method are highly dependent on the original injection region, injection time, and injection dosage.
Based in part on the failures described above, imaging vascular systems remains a continuing challenge. Current methods for diagnostic imaging of the vascular system are dependent on magnetic resonance or digital subtraction angiography. The main limitation of conventional angiography is its invasiveness, since the procedure often requires percutaneous vessel cannulation with the introduction of intravascular wires and devices.
Attempts have been made to perform molecular imaging to visualize the fundamental biological processes in developing the vascular system. The imaging of the vascular system requires the imaging reagents to selectively absorp on the surface of blood vessels, whose fluorescence would provide a sharp image of the vessel. The reagents also need to be non-toxic. One current imaging reagent for vascular systems utilizes quantum dots, but leakage of toxic cadmium ion poses a health concern. In addition, the quantum-dots-based imaging reagents require injection, and only allow a short time period for imaging (about 0.5 hour after injection of imaging dye).
Currently methods of protein detection also suffer from several drawbacks. The detection and discrimination of different proteins with high sensitivity and selectivity are very important for biological studies and clinical diagnosis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blotting is a current technique for protein analysis. The western blotting method has been widely used to separate, detect, and identify specific proteins in a complex mixture. However, in order to achieve the selective detection of a specific protein, time-consuming, multistep processes must be used. These processes also involve the use of expensive reagents and can require the protein transfer, blocking, and reaction with the primary and secondary antibodies, all followed by fluorescent detection of the bound antibodies. An essential step in western blotting is to stain the proteins following the separation by SDS-PAGE, where the post-stain washing is often necessary to remove the excess dyes. Therefore, it is highly desirable to develop new strategies that can facilitate protein visualization on a gel without going through a multiple steps process.
Among the known protein sensors, only few are capable of detecting proteins in gels. In addition, these dyes either exhibit low selectivity in responding to different proteins, or suffer from strong interference from SDS. The requirement and demand for improved staining can be seen from SYPRO Ruby, which is widely used for staining the proteins in SDS-PAGE. The standard process using SYPRO Ruby requires washing out the SDS, a long time for staining, and removing the excess dye. It is thus highly desirable to develop a simplified procedure that allows direct staining without the need for protein fixing, i.e. prior to staining, and washing out the non-bound staining reagent. Also, current staining methods generally display all proteins without discrimination.
Serum albumin is the most abundant protein in blood plasma and plays a key role in the disposition and transport of various endogenous ligands, fatty acids, and drugs. Some biological functions of serum albumin are associated with its natural hydrophobic pocket, which can be used to bind flavone dyes.
Thus, a need remains in the art for an improved biological imaging agent. A need also remains for a reliable and easy-to-use strategy for YSL imaging. A further need remains for an improved imaging reagent for displaying vascular systems.